Project Summary Colorectal cancer (CRC), the second leading cause of cancer deaths in the United States, is caused by mutations that disrupt orderly homeostasis of intestinal stem cells (ISCs). ISC and epithelial homeostasis require the maintenance of signaling gradients that limit ISCs to the crypt base and promote cell differentiation toward the top. BMP signaling forms one such essential gradient, with high BMP levels in the villus mesenchymal compartment and suppression of BMP via antagonists near the crypt bottom. Although BMP signals are generated and maintained in the underlying mesenchyme, it is unclear which cell types contribute toward maintaining the gradient. Using high resolution confocal microscopy and both bulk and single-cell RNA sequencing, my preliminary data reveal three PDGFRA+ mesenchymal populations that are anatomically and molecularly distinct. High-expressing PDGFRA cells, previously described as telocytes, lie closest to the epithelium, most abundantly at the crypt-villus junction, and express high levels of BMP ligands. The two other populations express low levels of PDGFRA and are anatomically farther from the epithelium than telocytes: CD81+PDGFRA-lo cells are found below crypts and express high levels of the BMP antagonist Gremlin1; CD81-PDGFRA-lo cells occur above crypts and lack Gremlin1 expression. In my preliminary co-culture experiments with isolated crypt epithelium and each of these purified cell types, CD81+ cells functionally replace all growth factor-supplemented media, CD81- cells support organoids minimally, and telocytes do not. Based on these findings, I hypothesize that CD81+PDGFRA-lo cells and telocytes together produce the BMP gradient that restrict ISCs to crypt bottoms and I propose to define the role of these novel cell populations in ISC homeostasis. First, I will test what signaling factors in CD81+ cells promote organoid growth. I will fully characterize CD81+ and CD81- cells in vivo to determine their full transcriptional profiles and use an established co-culture method to test which specific trophic factors promote organoid growth. Second, I will test whether telocytes and CD81+ cells maintain the BMP gradient. Expanding the established co-culture assay into in vitro ?sandwich? cultures that mimic the anatomic relationship of these cells in vivo, I will test if polarization of telocytes and CD81+ cells is responsible for the BMP gradient. Collectively, these studies have broad implications for intestinal biology and development of CRC.